According to figures released by the World Health Organization in December 2008 there are approximately 33 million people worldwide that are living with HIV infection, with 2.7 million newly reported infections and 2 million deaths yearly. Furthermore, the Public Health Agency of Canada reports a steady number of newly diagnosed cases over the past 12 years (HIV and AIDS in Canada: Surveillance Report to Dec. 31, 2008, Public Health Agency of Canada, 2009), with an estimated 58,000 Canadians living with HIV/AIDS. The scope of this global pandemic underscores the need for continued research efforts to find new avenues for prevention and treatment strategies.
While there is no current vaccine available to prevent HIV infection, for infected individuals there are a variety of anti-retroviral medications available that fall into four broad categories: Reverse transcriptase (RT) inhibitors, protease inhibitors, integrase inhibitors, viral entry inhibitors, and combinations thereof known as HAART (highly active antiretroviral therapy). While these therapies have undeniably improved the prognosis for HIV patients, there remain a number of drawbacks. These include a lack of complete viral suppression, as well as numerous, and sometimes serious, side effects (1). For these reasons intense research is focused on finding new avenues to combat the disease by developing drug-targeting strategies against key mediators of viral pathogenesis. For example, continued efforts are aimed at novel vaccine development strategies and recent attention has focused on the HIV Tat transcription factor as a drug target. This is due to its crucial role in a variety of viral functions including early RNA splicing, replication and transcription (2).
CAF and the CD8+ Anti-HIV Response
Upon infection with the HIV virus the host immune response is activated in an attempt to combat the infection and prevent the progression to AIDS. CD8+ T cells perform a crucial part of the host anti-HIV response in different ways. First, CD8+ cells from HIV infected individuals can recognize infected cells and respond by killing the cells lytically (CTL response). Typically, this response occurs during the initial phase of infection where a transient decrease in viremia can be seen. Also during the acute phase of infection, prior to antibody production, a further action of CD8+ T cells involves an innate, non-cytotoxic action, which results in a decrease of viral replication. This latter response is known as the CD8+ cell non-cytotoxic anti-HIV response (CNAR), and functions in a non-MHC restricted fashion (3, 4). While both of these activities are significant, the CNAR activity is of particular interest as it correlates with clinical status (5, 6). Specifically, long term HIV non-progressors, represented by infected but asymptomatic individuals who remain healthy without ever receiving anti-viral medication, display a heightened CNAR activity as compared to patients who display disease progression (7, 8).
CNAR is active against a wide variety of HIV strains and is able to suppress HIV replication in both CD4+ T cells as well as macrophages (9). Since its discovery in 1986, there has been keen interest by many research groups in understanding the molecular mechanism by which CNAR functions, however this remains incompletely understood. Several important studies have contributed to a partial characterization of CNAR.
It has been known for some time that the anti-HIV activity of CNAR results from the secretion of a proteinacious factor(s) from activated CD8+ T cells, and has been termed CAF for CD8+ anti-HIV factor (10). Interestingly, it has been shown that the secretion of an active HIV inhibitory CAF factor(s) is not limited to CD8+ T cells from HIV seropositive patients, but can also be detected in culture media from EBV specific T cell line (11). This result suggests that there may be a common or related CAF that could function to inhibit viral replication as a result of immune system activation.
Although several groups have identified proteins that display anti-HIV activity, none of these fulfill all the criteria that CAF is known to exhibit. Some examples of anti-HIV factors include alpha defensin, RANTES and other beta chemokines, the cell adhesion molecule VCAM, and bovine antithrombin III (12-15).
While the precise identity of CAF remains unknown, research by many groups has combined to reveal certain key features. Together with secretion from activated CD8+ T cells, studies have suggested that a proteolytic activity appears to be necessary for CAF activation since protease inhibitors can block CAF activity (16). To date, the responsible protease involved has not been identified. Mechanistically, it is known that CAF or a CAF-like activity can suppress viral replication by inhibiting HIV transcription driven by the viral LTR, but does not appear to function through inhibiting viral integration or reverse transcription (17-19). Further evidence suggests that the inhibition of viral transcription occurs via activation of the STAT1 signaling pathway (20). Given the observation that CAF activity functions through inhibition of viral LTR driven transcription, a mutational analysis of several transcription factor binding sites in the LTR has been performed (21). The results of this study indicate that CAF does not appear to rely on independent binding of NFAT, AP1, NF-κB or SP1 sites. With respect to viral transactivators, it is known that the HIV Tat transcription factor plays an essential role in viral replication through its interaction with the Tat transactivation response region (TAR). The TAR element is present both within the LTR as well as at the 5′ end of all viral transcripts where Tat interacts to activate transcription and promote efficient mRNA translation (22). The importance of the Tat/TAR system is highlighted by studies showing that impairing this interaction can dramatically inhibit HIV replication (23). The Tat/TAR system, perhaps together with other factors, is therefore a possible target for the effects of CAF.
Despite years of ongoing research in this area, there continues to be a need for new and improved medicines for treating and/or preventing HIV and AIDS. The present inventors have accordingly sought to identify new diagnostic and chemotherapeutic methods in this area by investigating the Tat/TAR system and potential CAF targets.